Derivatives of neomycin b and neomycin c

ABSTRACT

Derivatives of neomycin B and C have been prepared which possess substantially improved antibacterial activity. An example of such an agent is 1-(L-(-)- gamma -amino- Alpha -hydroxybutyryl)neomycin B (IVa, BB-K77).

United States Patent [1 1 Naito et al.

[111 3,860,574 1 Jan. 14, 1975 DERIVATIVES OF NEOMYCIN B AND NEOMYCIN C Inventors: Takayuki Naito; Susurnu Nakagawa;

Masahisa Oka, all of Tokyo, Japan Assignee: Bristol-Myers Company, New York,

Filed: June 27, 1973 Appl. No.: 374,191

Related US. Application Data Continuation-in-part of Ser. No. 312,452, Dec. 6, 1972, abandoned.

US. Cl... 260/210 NE, 260/210 AB, 424/180,

424/181 Int. Cl. C07c 95/04 Field of Search 260/210 AB, 210 NE Primary ExaminerJ0hnnie R. Brown Attorney, Agent, or Firm-Robert E. Havranek [57] ABSTRACT Derivatives of neomycin B and C have been prepared which possess substantially improved antibacterial activity. An example of such an agent is l-[L-()-'yamino-a-hydroxybutyryll-neomycin B [IVa, BB-K7 7].

13 Claims, N0 Drawings DERIVATIVES OF NEOMYCIN B AND NEOMYCIN C CROSS-REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of copending application Ser. No. 312,452, tiled Dec. 6, 1972, now abandoned.

BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to semisynthetic l-substituted derivatives of neomycin B and C, said compounds being-prepared by acylating the l-amino-function of neomycin B and C with a 'y-amino-a-hydroxylbutyryl, B-amino-a-hydroxypropionyl and B-amino-ahydroxyvaleryl moieties.

2. Description of the Prior Art The neomycins are known antibiotics described in Merck Index, 8th Edition, pp. 723-724. The compounds are fermented as a complex from which neomycin A, B and C have been isolated.

Neomycin B is a compound having the formula e e 6 III Neomycin C is a compound having the formula SUMMARY OF THE INVENTION The compound having the formula in which either R or R is CH NH and the other is hydrogen, in which R is L-()-'y-aminoahydroxybutyryl, L-( )-B-amino-a-hydroxypropionyl or L-()-8-amino-a-hydroxyvaleryl; or a nontoxic pharmaceutically acceptable acid addition salt thereof is a valuable antibacterial agent.

This invention relates to semi-synthetic derivatives of neomycin B or C, said compounds being known as l- [L-()-'y-amino-a-hydroxybutyryl] neomycin B or C (IVb or IVc), l-[L-()-B-amino-a-hydroxypropionyl] neomycin B or C (IVd or We) or 1-[L-( )-8-amino-ahydroxyvaleryl] neomycin B or C (IVf or lVg) and having the formula in which either R or R is CH NI-I and the other is hydrogen, R is L-()-y-amino-a-hydroxybutyryl, L- (-)-B-amino-a-hydroxypropionyl or L-()-8-amino-ahydroxyvaleryl; or a nontoxic pharmaceutically acceptable acid addition salt thereof.

For the purpose of this disclosure, the term nontoxic pharmaceutically acceptable acid addition salt shall mean a mono, di-, tri, tetra, penta or hexasalt formed by the interaction of one molecule of compound IV with l6 moles of a nontoxic, pharmaceutically acceptable acid. Included among these acids are acetic, hydrochloric, sulfuric, maleic, phosphoric, nitric, hydrobrornic, ascorbic, malic and citric acid, and those other acids commonly used to make salts of amine containing pharmaceuticals.

The compounds of the present invention are prepared by the following representative diagramatic scheme:

1.) Neomyci'n B 2. Compound III) or IR CH2-NH-C- or C N- (Bnzyloxycarbonyloxy) Succinimide N-Hydroxysuccinimide ester of A preferred embodiment of the present invention is and R is H. the compounds having the formula A most preferred embodiment is the compound of CH NEAR; formula V wherein R and R" are H and R is L-()-y- H 4 amino-a-hydroxybutyryl; or a nontoxic pharmaceuti- 5 cally acceptable acid addition salt thereof.

A most preferred embodiment is the compound of formula V wherein R and R are H and R is L-(l-B- amino-a-hydroxypropionylL or a nontoxic pharmaceutically acceptable acid addition salt thereof.

A most preferred embodiment is the compound of formula V wherein R and R are H and R is L-()-8- amino-a-hydroxyvaleryl; or a nontoxic pharmaceutically acceptable acid addition salt thereof.

Other most preferred embodiments are the sulfate,

hydrochloride, acetate. maleatc, citrate, uscorbatc, ni-

trate or phosphate salts of compound V.

Another most preferred embodiment is the mono and disulfate salts of compound V.

. The objectives of the present invention have been no achieved, by the provision according to the present invention of the process for the preparation of the compound having the formula Ho CH NH m4 W (re lm-R wherein both R and R are H or and R is H, L-()-y-amino-a-hydroxybutyryl, L-()-B- amino-a-hydroxypropionyl, L-()-5-amino-ahydroxyvaleryl, L-()-'y-benzyloxycarbonylamino-a- 40 hydroxybmflyli L" H3-benzyloxycarbony"ammo-1' in which either R or R is -Cl-l Nl-l and the other is hyhydroxyproplo y or drogen, in which R is L-()-7-amino-a-hydroxybutyryl, benzylioxycarbonylamino-a-hydroxyvaleryl; wherein R )-B- in -hydr0xypropionyl or L-()-8-aminoand R f" R must be other F a nontoxlc Phar' a-hydroxyvaleryl; or a nontoxic pharmaceutically ac .maceuncany acceptable addltfon Salt thereof ceptable acid addition salt thereof; which process com- Another preferred embodiment 15 the compound of prises the consecutive steps of formula V m which and R4 are A. acylating neomycin B or neomycin C with an acyl- 0 ating agent selected from the compounds having the CGH.,C formulas X CH -C-OH (or a. carbodiimide thereof) or t O ll . 3 CCH CH 'CO H (or a carbodiimide thereof),

in which R and R are alike or different and each is H, in which either R or R is -CH -NH-Y and the other is F. Cl, Br, N0 OH, (IOW UQ Y a y, X H, wherein Y is a radical of the formula I 0 CH3 0 I c on -c-o-.co N .5 I 2 is chloro, bromo or iodo, or a functionaliequivalent as an acylating agent; in a ratio of two moles or less of acylating agent per mole of neomycin B or C in a solvent, preferably selected from the group comprised of di- 20 v. methylformamide, dimethylacetamide, tetrahydroc 2 furan, dioxane, 1,2-dimethoxyethane, methanol, ethanol, water, acetone, pyridine, N-(lower)- alkylpiperidine, or mixtures thereof, but preferably 1:1 water-tetrahydrofuran at a temperature below 50 C. and preferably below 25 C., to produce the compound having the formula no -Y I -V v 3O @c-crr2-c2r -C-;

in which R and R are as defined above;

B. acylating compound II with an acylating agen 35 having the formula 3* it I-N (CH cH-cM v11:

0 in which n is an integer of l to 3 inclusive and W is .a

radical selected from the group consisting of fi but preferably CH2O-.C- M is a. radical 7 i 7t selected from the group comprising in which R and R are as above; in a ratio of at least 0.5 mole of compound VII per mole of compound 11, but preferably in a ratio of about 0.5 to about 1.4, and most preferably in a ratio of about 0.8 to about 1.1, in a solvent preferably'selected from the group comprising a mixture of water and ethyleneglyco] dimethyl ether, dioxane, dimethylacetamide, dimethylformamide, tetrahydrofuran, propyleneglycol dimethyl ether, or the like but preferably 1:1 water tetrahydrofuran to produce a compound of the formula R6 1 CH2 -o-cby hydrogenating compound [II with hydrogen in the presence of a metal catalyst, preferable selected from the group comprising palladium, platinum, Raney nickel, rhodium, ruthenium, and nickel, but preferably palladium, and most preferably palladium on charcoal, in a water-water miscible solvent system, preferably selected from the group comprising water and dioxane, tetrahydrofuran, ethyleneglycol dimethyl ether, propyleneglycol dimethyl ether, or the like, but preferably 1:1 water-tetrahydrofuran to produce the compound of formula V.

It should be apparent to those knowledgeable in the art that other agents can be used in the process above to acylate the amine functions of the intermediate compounds of the instant invention. This disclosure is meant to include all such acylating agents that produce 11 O 0-N -o-No .-o

.-0- -N but preferably labile amine blocking groups, said labile blocking groups commonly employed in synthesis of peptides. The labile blocking groups must be readily removable by methods commonly known in the art. Examples of said labile blocking groups and their removal can be found in the review of A. Kapoor, J. Pharm. Sciences 59, pp. 1-27 (1970). Functional equivalents as acylating agent for primary amine groups would include corresponding carboxylic chlorides, bromides, acid anhydrides, including mixed anhydrides and particularly the mixed anhydrides prepared from stronger acids such as the lower aliphatic monoesters of carbonic acid, of alkyl and aryl sulfonic acids and of more hindered acids such as diphenylacetic acid. In addition, an acid azide or an active ester of thioester (e.g., with p-nitrophenol, 2,4-dinitrophenol, thiophenol, thioacetic acid) may be used or the free acid itself may be coupled with the neomycin derivative (11) after first reacting said free acid with N,N-dimethylchloroforminiurn chloride [cf. Great Britain 1,008,170 and Novak and Weichet, Experientia XXI/6, 360 (1965] or by the use of enzymes or of an N,N'-carbonyldiimidazole or an N,N'- carbonylditriazole [cf. R. Buijile and H. G. Viehe, Angew, Chem., International Edition 3, 582 (1964)], or of a ketenimine reagent [cf. C. L. Stevens and M. E. Monk, J. Amer. Chem. Soc. 80, 4065 (1958)] or of an isoxazolium salt reagent [cf. R. B. Woodward, R. A. Olofson and H. Mayer, J. Amer. Chem. Soc., 83, 1010 (1961)]. Another equivalent of the acid chloride is a corresponding azolide, i.e., an amide of the corresponding acid whose amide nitrogen is a member of a quasiaromatic five membered ring containing at least two nitrogen atoms, i.e., imidazole, pyrazole, the triazoles, benzimidazole, benzotriazole and their substituted derivatives. As an example of the general method for the preparation of an azolide, N,N'- carbonyldiimidazole is reacted with a carboxylic acid in equimolar proportions at room temperature in tetrahydrofuran, chloroform, dimethylformamide or a similar inert solvent to form the carboxylic acid imidazolide in practically quantitative yield with liberation of carbon dioxide and one mole of imidazole. Dicarboxylic acids yield diimidazolides. The by-product, imidazole, precipitates and may be separate and the imidazolide isolated, but this is not essential. These reactions are wellknown in the art [cf. US. Pat. Nos. 3,079,315, 3,117,126 and 3,129,224 and British Pat. Nos. 932,644, 957,570 and 959,054).

Compounds IVb and 1V0, 1-[L-()-y-amino-ahydroxybutyryl)l-neornycin B (BB-K77) and 1-[L-()- y-amino-cz-hydroxybutyryll-neomycin C (BB-K88) respectively, possess excellent antibacterial activity that appears comparable or superior to their parents, neomycin B and C, respectively, against a wide spectrum of micro-organisms. Table 1 is illustrative of their comparative antibiotic activity.

Table I Continued In vitro Antimicrobial Activities of L-HABA Derivatives of Neomycins B and C Neomycin Neomycin MlC (meg/ml.) BB-K77 B FIB-K88 Mycuh. 607 1.6 0.8 1.6 3.1

do. KM-R 100 100 50 50 do. K M*,SM-R*** 100 100 50 50 do. phlei 0.4 0.4 0.8 1.6

do. ranae 0.8 0.8 1.6 3.1

KM-R is a kanamycin A resistant organism. GM-R" is a gentamicin resistant organism. SM-R'" is a streptomycin resistant organism. NM-R" is a neomycin resistant organism.

Compounds lVd (BB-K116), lVe (BB-K119), lVf EXAMPLE 1 (BB-K113) and lVg (BB-K132) were also assayed in vitro as compared to compounds lVb, lVc and neomycin B and C as shown below:

Compounds IV!) and [V generally appear more po-' tent against the test organisms than either IVd, IVe, IVf or 1Vg but all are still potent antimicrobial agents.

Compounds IV are valuable as anti-bacterial agents, nutritional supplements in animal feeds, therapeutic agents in poultry and animals, including man, and are especially valuable in the treatment of infectious diseases caused by Gram-positive and Gram-negative bacteria.

Compounds IV when administered orally are useful as an adjunctive treatment for preoperative sterilization of the bowel. Both aerobic and anaerobic flora which are susceptible to these drugs are reduced in the large intestine. When accompanied by adequate mechanical cleansing, they are useful in preparing for colonic surgery.

Compounds IV are effective in the treatment of systemic bacterial infections when administered parenterally in the dosage range of about 250 mg. to about 3000 mg. per day in divided doses three or four times a day. Generally the compounds are effective when administered at a dosage of about 5.0 to 7.5 mg./kg. of body weight every 12 hours.

Compounds of formula IV and the salts thereof are known to form monoand polyhydrates upon isolation from aqueous solvents. Accordingly, the hydrates so produced are considered an integral part of the instant invention.

Neomycin B and C both possess six amino moieties that are capable of acylation in the process for the preparation of the compounds of the instant invention. As such, it is not unusual to recover neomycin B and C derivatives other than those claimed from the process (see example 4).

PREPARATION OF NEOMYCIN B AND C Neomycins B and C were isolated by column chromatography of crude neomycin complex using Dowex 1-X2 according to the procedure of H. Maeher and C. P. Schaffner, Anal. Chem., 36 105 (1964).

Dowex l-X2 is a resin comprised of a polystyrene backbone to which is attached benzyltrimethylammonium chloride and 2% of added divinylbenzene as a cross-linking agent during polymerization of the poly styrene. It is supplied as beads. 1

Preparation of L-()-'y-Benzyloxycarbonylamino-a-hydroxybutyric Acid Vla) L-()-'y-amino-a-hydroxybutyric acid (7.4 g., 0.062 mole) was added to a solution of 5.2 g (0.13 mole) of sodium hydroxide in 50 ml. of water. To the stirred solution was added dropwise at 0-5 C. over a period of 0.5 hour, 11.7 g. (0.068 mole) of carbobenzoxy chloride and the mixture was continued to stir for one hour at the same temperature. The reaction mixture was washed with 50 ml. of ether, adjusted to pH 2 with dilute hydrochloric acid and extracted with four -ml. portions of ether. The ethereal extracts were combined, washed with a small amount of saturated sodium chloride solution, dried with anhydrous sodium sulfate and filtered. The filtrate was evaporated in vacuo and the resulting residue was crystallized from benzene to give 11.6 g. (74%) of colorless plates; melting point 78.579.5 C., [011 4.5 (c=2, CH Ol-1). Infrared (IR) [KBr] lR(KBr)yc=o 1740, 1690 cm. Nuclear Magnetic Resonance (NMR) (acetone-d 6 (in ppm from TMS) 2.0 (2H,m), 3.29 (2H,dd, J=6.7 and 12 Hz), 4.16 (1H,dd, J=4.5 and 8 Hz), 4.99 (2H,s), 6.2 (2H, broad), 7.21 (SI-Ls).

Anal. calcd. for C H NO C, 56.91; H, 5.97; N,

5.53. Found: C, 56.66; H, 5.97; N, 5.47

EXAMPLE 2 N-Hydroxysuccinimide ester of L-()-y-benzyloxycarbonylamino-a-hydroxybutyric Acid (Vlla) A solution of 10.6 g. (0.042 mole) of Vla and 4.8 g.-

(0.042 mole) of N-hydroxysuccinimide in 200 ml. of ethyl acetate was cooled to 0 C. and then 8.6 g. (0.042 mole) of dicyclohexylcarbodiimide was added. The mixture was kept overnight in a refrigerator. The dicyclohexylurea which separated was filtered off. and the filtrate was concentrated to about 50 ml. under reduced pressure to give colorless crystals of Vlla which were collected by filtration; 6.4 g., m.p. 12l-122.5 C. The filtrate was evaporated to dryness in vacuo and the crystalline residue was washed with 20 ml. of a benzene-n-hexane mixture to give an additional amount of Vlla. The total yield was 13.4 g. (92%). [01],, 1.5 (17 2, CHCl lR(KBr) 'y 1810, 1755, 1740, 1680 cm. NMR (acetone-d 8(in ppm from TMS) 2.0 (2H,m), 2.83 (4H,s), 3.37(2H,dd, J=6.5 and 12.5 Hz),

4.56(1H,m), 4.99(2H,s), 6.3(1H,broad), 7123 (H,s). (1. G. W. Anderson et al., H. Am. Chem. Soc., 86, 1839, (1964).)

Anal. calcd. for C H N O z C, 54.85; H, 5.18; N,

8.00. Found: C, 54.79, 54.70; H, 5.21, 5.20; N, 8.14, 8.12.

EXAMPLE 3 I Preparation of 6',6"'-Dibenzyloxycarbonylneomycin B To a stirred solution of 4.0 g. (6.5 m mole) of neomycin B in 200 ml. of 50% aqueous THF (tetrahydrofuran) was added 2.74 g. (11 m. mole) of N- benzyloxycarbonyloxysuccinimide at 5-10 C. The reaction mixture was stirred overnight at room temperature and evaporated in vacuo to remove the organic solvent. The aqueous solution was diluted with 100 ml. of water, washed with two 50-ml. portions of n-BuOH and stirred with ml. of CG-5O (NH, form) for two hours. The resin was collected by filtration, washed with deionized water and placed on the top of a column of CG-50 ion exchanger (NH form, 80 ml.), which was eluted successively with each one of 0.05 N NH OH, 0.1 N NH OH, 0.2 N NH OH and 0.3 N NH OH. The eluate was collected in -ml. fraction and monitored by ninhydrin test and TLC (thin layer chromatography) on silica gel plate (solvent system: MeOAc-n-PrOH-concentrate NH OH=45:105:60). Fractions 125 to 136 which showed a ninhydrin spot at Rf 0.49 were combined, concentrated in vacuo and freeze-dried to give 550 mg. (10%) of the desired product. m.p. 156158 C.; IR (infrared spectrum) (KBr) 3360, 1700, 1530 cm; NMR (nuclear magnetic resonance spectra) (DMSOd +D O) 4.70 (m, 1H), 4.97 (s, 4H) 5.05 (m,21-1), 7.22 (5,1011).

EXAMPLE 4 Preparation of 1[L-()-y-amino-a-hydroxybutyryl]-neomycin B (IVb) To a stirred solution of 500 mg. (0.566 m.mole) of 6,6"'-dibenzyloxycarbonylneomycin B in 20 ml. of

50% aqueous THF was added in one portion 180 mg. (0.566 m.mole) of N-hydroxysuccinimide ester of L- (-)-'y-amino-a-hydroxybutyric acid. The reaction mixture was stirred overnight at room temperature and hydrogenated with 1 g. of 10% palladium on charcoal for 3 hours at 50 psi. After removing the catalyst,-the filtrate was concentrated in vacuo to remove the organic solvent and the concentrate was treated with one ml. of CG-50 ion-exchange resin (NH, form) for 20 minutes. The resin was filtered, washed with water and puton the top ofa CG-50 column (NH.,*, 15 ml.). The column was eluted successively with each 500 ml. of 0.1 N, 0.3 N, 0.4 N and 0.5 N NH OH. The eluate was collected in 10-ml. fraction and grouped into the following cuts based on ninhydrin test, disc assay and TLC on slica gel plate (solvent system: CHCl MeOHconcentrated NH 0HH O-1:4:2:1). Each cut was evaporated in vacuo and the residue lyophilized.

Code No. M.p. (dec.) Rf* yc=o (KBr) BB-K77 l81184 0.19 1640 cm BB-K78 230 0.19 1630 BB-K79 222 0.19 1620 TLC: silica gel plate. CHCl,MeOH-28% NH.OHH,O (1:4:211)

The fraction BB-K77 was determined to be 1-[L-(-)- 'y-amino-a-hydroxybutyryl]-neomycin B, the desired product (lVb).

EXAMPLE 5 Preparation of 6',6"-Dibenzyloxycarbonyl-neomycin C (11c) To a stirred solution of 3.7 g. (6.03 m. mole) of neomycin C in 100 ml. of 50% aqueous THF was added 2.9 g. (12 m.mole) of N-benzyloxycarbonyloxysuccinimide at 5-10 C. The reaction mixture was stirred overnight at room temperature and evaporated in vacuo to remove the organic solvent. The aqueous solution was diluted with 100 ml. of water washed with two 50-ml. portions of n-BuOH and treated with 7 ml. of CG-50 (NH; form) for two hours. The resin was collected by filtration, washed with deionized water and placed on the top of a CG-50 column (NH; mm, 80 ml.), which was eluted successively with each 500 ml. of 0.1 N and 0.2 N NH OH. The eulate was collected in 10-m1. fraction and monitored by ninhydrin test and TLC on silica gel plate (solvent system: MeOAc-n-PrOH-28% NH OH=45:105:6O). Fractions 74-79 which showed a ninhydrin spot at Rf 0.49 were combined, concentrated in vacuo and lyophilized to give 210 mg. (4%) of the desired product; m.p. l41145 C.; IR (KBr) 3350, 1705, 1450 cm.

EXAMPLE 6 Preparation of l[L-()--y-amino-a-hydroxybutyryl]-neomycin C (We) To a stirred solution of 200 mg. (0.22 m.mole) of 6',- 6"-dibenzyloxycarbonylneomycin C in 20 ml. of 50% aqueous THF was added 77 mg. (0.22 m.mole) of N- hydroxysuccinimide ester of L-(-)-y-amino-ahydroxybutyric acid. The reaction mixture was stirred overnight, hydrogenated with mg. of 10% palladium charcoal for six hours at atmospheric pressure and filtered. The filtrate being concentrated in vacuo to remove the organic solvent, the resulting aqueous solution was treated with one ml. of CG-50 (NH, form). The resin was filtered washed with water and placed on the top of a CG-SO column (NH form. 4 ml.) The column was eluted successively with 200 ml. of0.1 N NH OH and 300 ml. of 0.3 N NH OH. The eluate was collected in 5-ml. fraction monitored by TLC on silica gel plate and disc assay (test organism: E. coli A20665). Fractions 58 to 60 (a ninhydrin spot at Rf 0.37) were combined,'evaporated in vacuo and lyophilized to give 7 mg. of neomycin C. Fractions 86 to 89 (a ninhydrin spot at Rf 0.15) gave 2 mg. of the desired product, BB-K88; m.p. -191C. (dec.); IR (KBr) Amberlite CG-50 is the tradename for the chromatographic grade of a weakly acidic cationic exchange resin of a carboxylic-polymethacrylic type.

EXAMPLE 7 Preparation of N-(Benzyloxycarbonyloxy)succinimide N-Hydroxysiccinimide (23 g., 0.2 mole) was dissolved in a solution of 9 g. (0.22 mole) of sodium hy-' droxide in 200 ml. of water. To the stirred solution was added dropwise 34 g. (0.2 mole) of carbobenzoxy chloride with water-cooling and then the mixture was stirred at room temperature overnight to separate the carbobenzoxy derivative which was collected by filtration, washed with water and air-dried. Yield 41.1 g. (82%). Recrystallization from benzene-n-hexane (:1) gave colorless prisms melting at 7879. (1. G. W. Anderson et al., J. Am. Chem. Soc., 86, 1839 (1964).

EXAMPLE 8 Preparation of L-()-y-amino-a-hydroxybutyric Acid from Ambutyrosin A or B or mixtures Thereof Ambutyrosin A (5.0 gm.) [U.S. Pat. No. 3,541,078, issued Nov. 17, 1970] was refluxed with 160 ml. of 0.5 N sodium hydroxide for one hour. The hydrolysate was neutralized with 6N HCl and chromatographed on a column of CG-SO (NH; type). The desired L-()-yamino-a-hydroxybutyric acid was isolated by developing the column with water and removing the water by freeze drying. The L-()y-amino-a-hydroxy-butyric acid is characterized as a crystalline material having a m.p. of 212.5214.5 C. [Column 2, lines 31-38, US. Pat. No. 3,541,078].

EXAMPLE 9 Preparation of L-()-amino-a-hydroxybutyric Acid from DLa-hydroxy-y-phthalimidobutyric Acid A. Dehydroabietylammonium phthalimidobutyrate To s solution of 25 g. (0.1 mole) of Z-hydroxy-yphthalimidobutyric acid in 200 ml. of ethanol was added a solution of 29 g. (0.1 mole) of dehydroabietylamine in 130 ml. of ethanol. The solution was shaken vigorously for a minute and stood at room temperature for five hours during which time fine needles crystallized out. The crystals were collected by filtration, washed with 50 ml. of ethanol and air-dried to obtain 30.1 g. (56%) of a diastereomer of the dehydroabietylamine salt. M.p. 9394 C. [04], (C.2.5, MeOH). Recrystallization from 300 ml of ethanol gave 23.2 g (43%) of the pure product. M.p. 9495C. [11],, 10.8 (C.2.5, MeOH). Further recrystallization did not change the melting point and the specific rotation. (1. Y. Satio etal. Tetrahedron Letters, 1970, 4863.)

Anal. calcd. for C H N O H O: C, 69.54; H, 8.02;

N, 5.07. Found: C, 69.58; H, 8.08; N, 5.07. B. L-(-)-y-amino-a-hydroxybutyric Acid L-a-hydroxy-y- To a solution of 1.5 g. (0.014 mole) of sodium carbonate in 40 ml. of water were added 5.3 g. (0.01 mole) of dehydroabietylammonium L-a-hydroxy-yphthalimidobutyrate and 60 ml. of ether. The mixture was shaken vigorously until all of the solid had dissolved. The ether layer was separated. The aqueous solution was washed twice with 30-ml. portions of ether and evaporated to 15 ml. under reduced pressure. To the concentrate was added 10 ml. of concentrated hydrochloric acid and the mixture was refluxed for ten hours. After cooling, separated phthalic acid was removed by filtration. The filtrate was evaporated under reduced pressure. The residue was dissolved in 10 ml. of water and the solution was evaporated to dryness. This operation was repeated twice to remove excess hydrochloric acid. The residual syrup was dissolved in 10 ml. of water and filtered to remove a small amount of insoluble phthalic acid. The filtrate was adsorbed on a column of IR-120 (H*, 1 cm. X 35 cm.), the solumn was washed with 300 ml. of water and eluted with l N ammonium hydroxide solution. The eluate was collected in 15-ml. fraction. The ninhydrin positive fractions 10 to 16 were combined and evaporated under reduced pressure to give a syrup which crystallized gradually. The crystals were triturated with ethanol, filtered and dried in a vacuum desiccator to give 0.78 g. (66%) of L-()-'y-amino-a-hydroxybutyric acid. M.p. 206207 C. [01],, 29 (C, 2.5, 11 0). The IR spectrum were identical with the authentic sample which was obtained from ambutyrosin.

EXAMPLE 10 Preparation of the Monosulfate Salt of l-[L-()-y-amino a-hydroxybutyryl]neomycin B or C One mole of 1-[L-()-y-amino-a-hydroxybutyryl]- neomycin B or C is dissolved in 1 to 3 liters of water. The solution is filtered to remove any undissolved solids. To the chilled and stirred solution is added one mole of sulfuric acid dissolved in 500 ml. of water. The mixture is allowed to stir for 30 minutes, following which cold ethanol is added to the mixture till precipitation occurs. The solids are collected by filtration and are determined to be the desired monosulfate salt.

EXAMPLE 1 1 Preparation of the Disulfate Salt of 1-[L-()- amino-a-hydroxybutyryl]-neomycin B or C One mole of 1-[L-()y-amino-a-hydroxybutyryl]- neomycin B or C is dissolved in 1 to 3 liters of water. The solution is filtered to remove any undissolved solids. To the chilled and stirred solution is added two moles of sulfuric acid dissolved in ml. of water. The mixture is allowed to stir for 30 minutes, following which cold ethanol is added to the mixture until precipitation occurs. The solids are collected by filtration and are determined to be the desired disulfate salt.

EXAMPLE 12 Preparation of L-B-Benzyloxycarbonylamino-a-hydroxypropionic Acid (VIb) L-B-Amino-a-hydroxypropionic acid* (8.2 g., 0.078

mole) was dissolved in a solution of 6.56 g. (0.0164

mole) of sodium hydroxide and in 60 m1. of water. To the stirred solution was added dropwise 14.7 g. (0.086 mole) of carbobenzoxy chloride below 5 C. The mixture was stirred for an hour at room temperature, washed with 60 ml. of ether and adjusted to pH 2 with dilute HCl. The precipitate was collected by filtration, washed with water and air-dried to give 9.65 g. (52%) of Vlb. The filtrate was extracted with five 100-ml. portions of ether. The ethereal solution was washed with water, dried over sodium sulfate and evaporated to dry- 1 ness in vacuo to give additional 2.0 g. (11%) of Vlh. A total of 11.65 g. of Vlb. was crystallized from 500 ml. of benzeneethyl acetate (4:1) to give 936 g. (50%) of pure Vlb. m.p. 128.5-l29.5 C. Infrared (IR) (KBr) y =O 1745, 1690 cm. [111 +2.9 (c5.0, MeOH). Nuclear Magnetic Resonance Spectra ([NMR (DMSO- d 8(in ppm) 3.05-3.45 (2H, m, CH N), 4.05 (1H, dd, -OCHCO), 5.03 (2H, s, CH Ar) 7.18 (1H, broad, NH), 7.36 (5H, s, ring H).

- benzyloxycarbonylamino-a-hydroxypropionic (K. Frcudcnhcrg, Ben, 47.21)27( l 914).) C:

Anal. calcd. for C H NO C, 55.23; H, 5.48; N,

5.86. Found: C, 55.34; H, 5.49; N, 5.87.

EXAMPLE l3 N-Hydroxysuccinimide Ester of L-B-benzyloxycarbonylaminoa-hydroxypropionic Acid (Vllb) EXAMPLE 14 Preparation of 1-[L-()-B-amino-a-hydroxypropionyl]neomycin B (lVd, BB-K116) To a stirred solution of 2.44 g (2.73 m moles) of 6, 6"'-N-dibenzyloxycarbonylneomycin B (11b) in 60 ml of 33% aqueous tetrahydrofuran (THF) was added a solution of the activated ester (VIlb) in 20 ml of THF prepared from 588 mg (2.46 m moles) of L-B- acid. The reaction mixture was stirred overnight and then hydrogenated overnight with 1.1 g of palladium on charcoal in a Parr hydrogenator at 45 psi and room temperature. The catalyst was filtered off and washed with water. The filtrate and washings were evaporated to remove most of the organic solvent. The resulting aqueous solution was passed through a column of amberlite CG-50 (NH +,55 ml), which was eluted with 400 ml of water and each 1 L of 0.1 N NH OH, 0.3 N NH OH and 0.5 N NH CH. The eluate was collected in -ml fraction from tube nos 1 to 140 and then in 10-ml fractions, and cut into the following appropriate fractions on the bases of TLC on slica gel plate (S-1 10, ninhydrin) and disk assay using B. subtilis PC] 219 and P. aeruginosa A 9843. Each fraction was evaporated in vacuo and lyophilized.

Fraction Tube No. NH.OH Weight Identification 1 70 74 0.3 N 206 mg(12%) neomycin B 2 201-202 0.5 N 236 mg(14%) BB-K 116, the

desired compound 3 217-224 0.5 N 22. mg 1.4%) BB-K 117,

a position isomer Physico-chemical properties:

Code No. Mp (dec) IR (KBr): -yC=O Rf (S1 10, ninhydrin) BB-K 116 195l97 1640 cm" 0.43 BB-K 117 20l-203 1640 cm 0.23

Microanalysis of BB-K 116: Calcd for C H N O JAH CO C. 43.46; H, 7.15; N. 13.38. Found: C.43.4l; H. 7.57; N. 12.88.

Both 1 16 and BB-K 117 gave BB-K B-amino-a-hydroxypropionic acid and neomycin B on hydrolysis with 0.5 N NaOH at C.

EXAMPLE 15 Preparation of l-N-[L()-,B-Amino-a-hydroxypropionyl]neomycin C (We, BB-K119).

To a stirred solution of 2.7 g (3 m moles) of6 .6- N-dibenzyloxycarbonylineomycin C (11c) in 60 ml of 33%Bfiii6hs THF was added a solution of the activated ester'(Vllb) in 20 ml of THF which was preparedfrom 645 mg (2.7 m moles) of L-fibenzyloxycarbonylamino-a-hydroxypropionic acid. The mixture was stirred overnight and evaporated in vacuo to remove the organic solvent. The resulting aqueous solution was diluted with 50 ml of water and extracted three l00-ml portions of n-butanol. The butanol extracts were evaporated to dryness. The residue was dissolved in 50 ml of 40% aqueous THF. The solution was hydrogenated overnight with 1.1 g of 10% palladium on charcoal in a Parr hydrogenator at 45 psi and room temperature. The catalyst was removed by filtration and washed with water. The filtrate and washings were evaporated in vacuo to remove most of the organic solvent. The resulting aqueous solution was passed through a column of amberlite CG-SO (NHJ, 55 ml), which was irrigated successively with 400 ml of water, 400 ml of 0.1 N. 1 L of 0.3 N and L L of 0.5 N NHqOH. The eluate was collected in 20-ml fraction and divided into the following appropriate fractions on the bases of TLC on silica gel plate (S-110. ninhydrin) and disk assay using B. sublilis PCl 219 and E. coli K-12 A 20665. Each fraction was evaporated in vacuo and lyophilized.

C, 40.73; H. 6.71; N. 11.87 Found: C. 40.71; H. 6.73; N. 12.08.

BB-K 119 and BB-K 120 gave neomycin C and B-amino-a-hydroxypropionic acid by hydrolysis with 0.5 N NaOH.

EXAMPLE 16 Preparation of L-(S-Benzyloxycarbonylamino-ot-hydroxyvaleric acid (Vlc) To a stirred solution of 400 mg (3.0 m moles) of L-8-amino-a-hydroxyvaleric acid* and 250 mg (6.5 m moles) of sodium hydroxide in 25 ml of water was added dropwise 580 mg (3.3 m moles) of carbobenzoxy chloride over a period of 30 minutes at -5C. The mixture was stirred for an hour at -15C, washed with 25 ml of ether, adjusted to pH 2 with hydrochloric acid and extracted with three 30-ml portions of ether. The combined ethereal solution was shaken with ml of a saturated sodium chloride solution, dried over an hydrous sodium sulfate and evaporated in vacuo to give crystals which were recrystallized from benzene to yield 631 mg (78%) of Vlc, mp 110-l11C.; infrared spectrum [lR(KBr)]: 3460, 3350, 1725, 1685, 1535, 1280, 730, 690cm. Nuclear magnetic resonance spectrum [NMR(acet0ne-d )]8 (in ppm) 1.70 (4H, m) 4.14 (2H, q, J=4.5Hz), 4.19(1H, m), 4.82(2H, s), 6.2(3H, broad), 7.25 (5H, s). [a],, 1.6 (c 10, MeOH),

Anal. Calcd for C H NO C. 58.42; H. 6.41; N,

Found: C, 58.36; H, 6.50; N, 5.27. (*S. Ohshiro et a1., Yakugaku Zasshi, 87, 1184 (1967).)

EXAMPLE l7 N-Hydroxysuccinimide ester of L-S-benzyloxycarbonylamino-a-hydroxyvaleric acid (VIIc) To a stirred and chilled solution of 535 mg (2.0 m moles) of Vlc and 230 mg (2.0 m moles) of N- hydroxysuccinimide in 55 ml of ethyl acetate was added 412 mg (2.0 m moles) of N, Ndicyclohexylcarbodiimide (DCC). The mixture was stirred for 3 hours at room temperature and filtered to remove precipitated N, N'-dicyclohexylurea. The filtrate was evaporated in vacuo to yield 780 mg (100%) of viscous syrup (Vllc). 1R(Neat):'y 1810, 1785, 1725 cm- EXAMPLE 18 Preparation of 1-N-[L()-8-Amino-a-hydroxyvaleryl]neomycin B, (IVf, BB-K 113) To a stirred solution of 2.7 g (3.0 m moles) of 6, 6"- N-dibenzyloxycarbonylneomycin B (11b) in 40 ml of 25% aqueous THF prepared from 724 mg (2.7 m moles) of L-8-benzyloxycarbonylamino-ozhydroxyvale'ric acid. The reaction mixture was stirred for 4 hours at room temperature and then hydrogenated overnight with 1.1 g of 10% palladium on charcoal in a Parr hydrogenator at 45 psi and room temperature. The catalyst was filtered off and washed with water. The filtrate and washings were combined and evaporated in vacuo to remove most of the organic solvent. The resultant aqueous solution was adsorbed on a column of Amberlite CG-50(NH 55 ml), which was irrigated successively with 200 ml of water, 200ml of 0.1 N, 900 ml of 0.3 N, 1.9 L of 0.5 N and 2 L of 1.0 N NH OH. The eluate was collected in 20-ml fraction from tube nos 1 to 20, then in -ml fraction and cut into the following appropriate fractions on the bases of TLC on a silica gel plate (S-110, ninhydrin) and disk assay by use of B. sublilis PC! 219 and P. aeruginosa A 9843.

-('ontint|cd Fraction Tube Nov NH,OH Weight Identification 3 146-164 0.5 N 42 mg12% llll-K 112.11

position isomer 4 234-271 0.5 N 243 mg111'/r) BB-K 11.1. the desired compound 5 300-311 1.0 N 366 mg( 15%) llll-K 114.

diztcylntcd compound 6 340-365 1.0 N 4) mg(2'% 1 111l-K 115.

111;1L"\1\lt:t1 compound Physico-chemical data:

Code No. Mp (dec) 1R1K111'l11 U RI S-1 10 BB-K 111 184-186(' 1640 cm 0.35 BB-K 112 195-197C 1640 cm 0 3S BB-K 113 190-197C 1640 ctn 0 30 BB-K 114 191-193C 1(140em 0.211 BB-K 115 192-194C 1640 cm 1 0.20

Microanalyses:

Calc'd for C ,,H N O, .2H CO C. 42.20; H 6.97;N.11.48.

BB-K 111: Found: C, 42.02; H, 6.95; N,

BB-K 112: Found: C, 42.49; H. 6.78; N,

BB-K 113: Found: C, 42.50; H. 6.67; N.

11.21. Calcd for C H N O 3/2H CO C, 44.18; H.

H, 7.20; N, 11.95. BB-K 114: Found: C. 44.08; H, 7.1); N,

12.01. Calcd for C H N O,,.2H C();,.H O: (142.5); H

7.15; N. 11.35. BB-K 115: Found: 42.53; H, 6.9); N,

All these BB-K compounds gave neotnycin 11 and 8-amino-a-hydroxyvaleric acid on hydrolysis with 0.5 N NaOH at 100C for 1 hour.

EXAMPLE 19 Preparation of 1-N-[L()-8-Amino-a-hydroxyvaleryllneomycin C (1Vg, BB-K 132) To a stirred solution of 2.7 g (3 m moles) of 6, 6"- N-dibenzyloxycarbonylneomycin C (He) in 60 ml of 33% aqueous THF was added a solution of the activated ester (Vllc) in 20 m1 of THF which was prepared from 724 mg (2.7 m moles) o1- L-8- benzyloxycarbonylamino-a-hydroxyvaleric acid. The mixture was stirred overnight at room temperature and then hydrogenated overnight with 1.1 g of 10/1 palladium on charcoal in a Parr hydrogenator at 45 psi and room temperature. The catalyst was removed by filtration' and washed with water. The filtrate and washings were evaporated in vacuo to remove most of the organic solvent. The resulting aqueous solution was passed through a column of amberlite CG-50 (NH 55 ml), which was irrigated successively with m1 of water, 260 m1 of 0.1 N, 1.3 L of 0.3 N, 800 ml of 0.5 N and finally l L of 1.0 N NH OH. The eluate was collectcd in 20-ml fraction and cut into the following appropriate fractions on the bases of TLC on silica gel plate (S-1 10, ninhydrin) and disk assay using B. .vuhrilix PCl 219 and E. coli K-12 A20665. Each fraction was evaporated in vacuo and lyophilized.

. Fraction Tube No. NH OH Weight Identity 1 32-42 0.3 N 474 mg(26%) neomycin C BB-K 13o. position isomer 2 70-81 0.3 N 54 mg(3%) 3 85-91 0.5 N 47 mg(27r) BB-K 131.

' position isomer 4 100-118 0.5 N 194 mg( 10%) BB-K 132,

the desired compound 5 127-l30 1.0 N 178 mg(97r) BB-K 133.

diacylated compound crude BB-K 134 diaeylated compound 6 147-159 1.0 N 49 mg Rechrematography of fraction 6 (49 mg) on amberlite CG-50 (NH., ,5 m1) afforded 26 mg (14%) of BB-K 134.

Physico-chemical data:

Code No. Mp(dec)- 1R (KBr):yC=O Rf (S-1 10. v ninhydrin) BB-K 130 201-202C 1640 cm 0.18 BB-K 131 190-202C 1640 cm 0.17 BB'K 132 195-196C 1640 cm 0.15 BB-K 133 201202C 1640 cm 0.11 BB-K 134 189-190C 1640 cm" 0.07

Microanalyses:

BB-K 130: Found: C, 41.33; H, 6.59; N, 10.94.

BBK 131: Found: C, 40.84; H, 6.58; N,

38-14 132: Found: C, 42.75; H. 6.97; N. 11.60.

Calcd for C H N O .2H CO .H O: C, 42.59; H,

BB-K 133: Found: C, 42.64; H. 7.08; N. 11.60.

BB-K 134: Found: C, 42.48; H. 6.60; N. 10.82

5 Ho c3 11 R cu nil-R v wherein both R" and R are H or R is H, L-()-'y-amin'o-a-hydroxybutyryl, 1,-()-B- amino-a-hydroxypropionyl, L-()-5-umin0-uhydroxyvaleryl, L-()-'y-benzyloxycarbony1amino-a hydroxybutyryl, L-()-B-benzyloxyearbonyl-amino-ahydroxypropionyl, or L-()-0- benzyloxycarbonylamino-a-hydroxyvaleryl, wherein R and R or Rmust be other than H; or a non-toxic pharmaceutically acceptable acid addition salt thereof.

2. A compound of claim 1 having theformula wherein both R and R are H or C H -CH eO-C I R is H, L-()-y-amino-a-hydroxybutyryl, 1.-()-B- amino-a-hydroxypropionyl, L-()-6-an 1ino-ahydroxyvaleryl, L-()-y-benzy1oxycarhonylamino-ahydroxybutyryl, L-()-B-benzyloxycarbonyl-amino-ahydroxypropionyl, or L-()-5-ben7.yloxycarbonylamino-a-hydroxyvaleryl, wherein R and R" or R must be other than H; or a nontoxic pharmaceutically acceptable acid addition salt thereof.

3. The compound of claim 2 wherein R and R are and R is hydroxybutyryl,

4. The compound of claim 2 wherein R and R are H and R is L-()-'y-amino-a-hydroxybutyryl, L-()B- aminou-hydroxypropionyl or L-()-8-amino-ahydroxyvaleryl; or a nontoxic pharmaceutically acid addition salt thereof.

5. The compound of claim 4 wherein R and R are H and R is L-()-y-amino-a-hydroxybutyryl; or the monoor disulfate salt thereof.

6. The compound of claim 4 wherein R and R are H and R is L-()-B-amino-a-hydroxypropionyl; or the monoor disulfate salt thereof.

7. The compound of claim 4 wherein R and R are H and R is L-()-8-amino-a-hydroxyvaleryl; or the monoor disulfate salt thereof.

8. The compound of claim 1 having the formula wherein both R and R are H or R is H, L-()-'y-amino-whydroxybutyryl, L-()- B- amino-a-hydroxypropionyl, L-()-8-amino-ahydroxyvaleryl, L-()-'y-benzyloxycarbonylamino-ahydroxybutyryl, L-()-B-benzyloxycarbonylamino-ahydroxypropionyl, .or L-()-8- benzyloxycarbonylamino-a-hydroxyvaleryl, wherein R and R or R must be other than H; ora nontoxic pharmaceutically acceptable acid addition salt thereof.

9. The compound of claim 8 wherein R and R are C6H5CH2-'O-C- and R is L-()-y-benzyloxycarbonylamino-ahydroxybutyryl, L-()-B-benzyloxycarbonylamino-ahydroxypropionyl or L()-8- benzyloxycarbonylamino-a-hydroxyvaleryl.

10. The compound of claim 8 wherein R and R" are H and R isL-()y-amino-a-hydroxyhutyryl, L-()-B- amino-a-hydroxypropionyl or L-()-8-amino-a hydroxyvaleryl; or a nontoxic pharmaceutically acid addition salt thereof.

11. The compound ofclaim 8 wherein R and R" are H and R is L-()-y-amino-a-hydroxybutyryl; or the monoor disulfate salt thereof.

12. The compound of claim 8 wherein R and R" are H and R is L-()-B-amino-a-hydroxypropionyl; or the monoor disulfate salt thereof.

13. The compound of claim 8 wherein R and R zlrc H and R is L-( )-8-amino-a-hydroxyvalcryl; or the monodisulfate salt thereof. 

2. A compound of claim 1 having the formula
 3. The compound of claim 2 wherein R3 and R4 are
 4. The compound of claim 2 wherein R3 and R4 are H and R is L-(-)- gamma -amino- Alpha -hydroxybutyryl, L-(-) Beta -amino- Alpha -hydroxypropionyl or L-(-)- delta -amino- Alpha -hydroxyvaleryl; or a nontoxic pharmaceutically acid addition salt thereof.
 5. The compound of claim 4 wherein R3 and R4 are H and R is L-(-)- gamma -amino- Alpha -hydroxybutyryl; or the mono- or disulfate salt thereof.
 6. The compound of claim 4 wherein R3 and R4 are H and R is L-(-)- Beta -amino- Alpha -hydroxypropionyl; or the mono- or disulfate salt thereof.
 7. The compound of claim 4 wherein R3 and R4 are H and R is L-(-)- delta -amino- Alpha -hydroxyvaleryl; or the mono- or disulfate salt thereof.
 8. The compound of claim 1 having the formula
 9. The compound of claim 8 wherein R3 and R4 are
 10. The compound of claim 8 wherein R3 and R4 are H and R is L-(-)- gamma -amino- Alpha -hydroxybutyryl, L-(-)- Beta -amino-Alpha -hydroxypropionyl or L-(-)- delta -amino- Alpha -hydroxyvaleryl; or a nontoxic pharmaceutically acid addition salt thereof.
 11. The compound of claim 8 wherein R3 and R4 are H and R is L-(-)- gamma -amino- Alpha -hydroxybutyryl; or the mono- or disulfate salt thereof.
 12. The compound of claim 8 wherein R3 and R4 are H and R is L-(-)- Beta -amino- Alpha -hydroxypropionyl; or the mono- or disulfate salt thereof.
 13. The compound of claim 8 wherein R3 and R4 are H and R is L-(-)- delta -amino- Alpha -hydroxyvaleryl; or the mono- disulfate salt thereof. 